Abstract Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infections in infants, and causes severe respiratory morbidity and mortality in the elderly, for which no effective treatment or vaccine is currently available. RSV infections have also been linked to the development and severity of asthma. Given the high annual incidence and wide range in the severity of illness caused by RSV, a biomarker capable of accurately and consistently predicting disease severity and post-acute outcomes of RSV infection is sorely needed. Previous studies have shown elevated neurotrophins, leukotrienes, and cytokines in RSV bronchiolitis as potential biomarkers; however, no mechanism-based biomarkers of RSV infection have yet been validated for diagnostic and/or prognostic purposes. In this study, we will dissect the role of post-translationally modified High Mobility Group Box-1 (HMGB1) in disease severity, and its secretion in activation of immune cells to promote inflammatory responses, and will investigate whether HMGB1 and its associated signaling pathways represent valid, prognostic biomarkers of RSV infection. HMGB1 is a multifunctional protein that serves as both a DNA regulatory protein and an extracellular cytokine signaling molecule that promotes inflammation as a damage-associated molecular pattern. The precise extracellular secretion mechanism for HMGB1 is not fully understood, but it is secreted after extensive post-translational modifications (PTMs). Recent studies have highlighted the importance of PTMs in HMGB1 function and release mechanisms. Specifically, secreted HMGB1 has been shown to undergo extensive PTMs such as acetylation, phosphorylation, glycosylation, and methylation that promote active transport of HMGB1 from the nucleus to the extracellular space. Our preliminary studies demonstrated that RSV infection hyperacetylates/phosphorylates HMGB1, thus leading to significant active release of HMGB1 from airway epithelial cells, and that secreted HMGB1 activates primary human monocytes to promote the inflammatory response. Based on our findings, we hypothesize that upon RSV infection, extensive PTMs of HMGB1, including hyperacetylation of lysine residues and serine phosphorylation within the nuclear localization sequence sites, promote HMGB1 mobilization from the nucleus to the cytoplasm and its subsequent extracellular release in airway epithelial cells, and that secreted HMGB1 activates signaling cascades to promote inflammation. Aim 1 will determine the role of HMGB1 PTMs in its extracellular release, immune cell activation and proinflammatory signaling, and test whether HMGB1 deacetylation through deacetylase enzyme SIRT 1 activation would inhibit RSV-induced HMGB1 acetylation and its proinflammatory activity. Aim 2 will determine whether distinct HMGB1 expression and/or PTM profile(s) at the airway mucosa can discriminate between infants with different severities of illness after naturally- acquired RSV infections. The outcome of these studies will have important therapeutic implications not only for RSV infection, but also for other respiratory diseases that affect human population of all age.